Yasushi Kaburagi

Insulin signalling and insulin actions in the muscles and livers of insulin-resistant, insulin receptor substrate 1-deficient mice.

We and others recently generated mice with a targeted disruption of the insulin receptor substrate 1 (IRS-1) gene and demonstrated that they exhibited growth retardation and had resistance to the glucose-lowering effect of insulin. Insulin initiates its biological effects by activating at least two major signalling pathways, one involving phosphatidylinositol 3-kinase (PI3-kinase) and the other involving a ras/mitogen-activated protein kinase (MAP kinase) cascade. In this study, we investigated the roles of IRS-1 and IRS-2 in the biological action in the physiological target organs of insulin by comparing the effects of insulin in wild-type and IRS-1-deficient mice. In muscles from IRS-1-deficient mice, the responses to insulin-induced PI3-kinase activation, glucose transport, p70 S6 kinase and MAP kinase activation, mRNA translation, and protein synthesis were significantly impaired compared with those in wild-type mice. Insulin-induced protein synthesis was both wortmannin sensitive and insensitive in wild-type and IRS-1 deficient mice. However, in another target organ, the liver, the responses to insulin-induced PI3-kinase and MAP kinase activation were not significantly reduced. The amount of tyrosine-phosphorylated IRS-2 (in IRS-1-deficient mice) was roughly equal to that of IRS-1 (in wild-type mice) in the liver, whereas it only 20 to 30% of that of IRS-1 in the muscles. In conclusion, (i) IRS-1 plays central roles in two major biological actions of insulin in muscles, glucose transport and protein synthesis; (ii) the insulin resistance of IRS-1-deficient mice is mainly due to resistance in the muscles; and (iii) the degree of compensation for IRS-1 deficiency appears to be correlated with the amount of tyrosine-phosphorylated IRS-2 (in IRS-1-deficient mice) relative to that of IRS-1 (in wild-type mice).

Annexin II Is a Novel Player in Insulin Signal Transduction POSSIBLE ASSOCIATION BETWEEN ANNEXIN II PHOSPHORYLATION AND INSULIN RECEPTOR INTERNALIZATION

Annexin II is a Ca2+-, phospholipid-, and actin- binding protein that was implicated in the regulation of vesicular traffic and endosome fusion. It is a known substrate for protein kinases including the platelet-derived growth factor receptor, src protein-tyrosine kinase, and protein kinase C. In the present study we investigated the possible involvement of annexin II in insulin signal transduction. Phosphorylation of annexin II in response to insulin treatment of intact Chinese hamster ovary (CHO)-T cells was detected by 5 min and reached maximal levels after a 2-3-h incubation with the hormone. However, unlike other receptor substrates, annexin II failed to undergo insulin-induced Tyr phosphorylation under conditions where receptor internalization was inhibited. This was evident in CHO cells, overexpressing the insulin receptor, in which internalization was inhibited either by tyrosine kinase inhibitors or by lowering the temperature to 4°C, and in CHO cells overexpressing various insulin receptor mutants in which normal internalization was impaired. Hence, Tyr phosphorylation of annexin II could be part of the internalization and sorting mechanism of the insulin receptor.

Role of insulin receptor substrate-1 and pp60 in the regulation of insulin-induced glucose transport and GLUT4 translocation in primary adipocytes.

In muscle and fat, glucose transport occurs through the translocation of GLUT4 from an intracellular pool to the cell surface. Phosphatidylinositol (PI) 3-kinase has been shown to be required in this process. Insulin is thought to activate this enzyme by stimulating its association with tyrosine-phosphorylated proteins such as insulin receptor substrate (IRS)-1, IRS-2, Grb2-associated binder-1, and pp60. To study the role of these endogenous substrates in glucose transport, we analyzed adipocytes from IRS-1 null mice that we previously generated (Tamemoto, H., Kadowaki, T., Tobe, K., Yagi, T., Sakura, H., Hayakawa, T., Terauchi, Y., Ueki, K., Kaburagi, Y., Satoh, S., Sekihara, H., Yoshioka, S., Horikoshi, H., Furuta, Y., Ikawa, Y., Kasuga, M., Yazaki Y., and Aizawa S. (1994) Nature 372, 182–186). In adipocytes from these mice, we showed that: 1) insulin-induced PI 3-kinase activity in the antiphosphotyrosine immunoprecipitates was 54% of wild-type; 2) pp60 was the major tyrosine-phosphorylated protein that associated with PI 3-kinase, whereas tyrosine phosphorylaion of IRS-2 as well as its association with this enzyme was almost undetectable; and 3) glucose transport and GLUT4 translocation at maximal insulin stimulation were decreased to 52 and 68% of those from wild-type. These data suggest that both IRS-1 and pp60 play a major role in insulin-induced glucose transport in adipocytes, and that pp60 is predominantly involved in regulating this process in the absence of IRS-1.

Csk Enhances Insulin-Stimulated Dephosphorylation of Focal Adhesion Proteins

Insulin has pleiotropic effects on the regulation of cell physiology through binding to its receptor. The wide variety of tyrosine phosphorylation motifs of insulin receptor substrate 1 (IRS-1), a substrate for the activated insulin receptor tyrosine kinase, may account for the multiple functions of insulin. Recent studies have shown that activation of the insulin receptor leads to the regulation of focal adhesion proteins, such as a dephosphorylation of focal adhesion kinase (pp125FAK). We show here that C-terminal Src kinase (Csk), which phosphorylates C-terminal tyrosine residues of Src family protein tyrosine kinases and suppresses their kinase activities, is involved in this insulin-stimulated dephosphorylation of focal adhesion proteins. We demonstrated that the overexpression of Csk enhanced and prolonged the insulin-induced dephosphorylation of pp125FAK. Another focal adhesion protein, paxillin, was also dephosphorylated upon insulin stimulation, and a kinase-negative mutant of Csk was able to inhibit the insulin-induced dephosphorylation of pp125FAK and paxillin. Although we have shown that the Csk Src homology 2 domain can bind to several tyrosine-phosphorylated proteins, including pp125FAK and paxillin, a majority of protein which bound to Csk was IRS-1 when cells were stimulated by insulin. Our data also indicated that tyrosine phosphorylation levels of IRS-1 appear to be paralleled by the dephosphorylation of the focal adhesion proteins. We therefore propose that the kinase activity of Csk, through the insulin-induced complex formation of Csk with IRS-1, is involved in insulins regulation of the phosphorylation levels of the focal adhesion proteins, possibly through inactivation of the kinase activity of c-Src family kinases.

Cytoskeletal reorganization induced by insulin: involvement of Grb2/Ash, Ras and phosphatidylinositol 3-kinase signalling

Background:u2002 Cytoskeletal reorganization is important for a wide variety of insulin‐mediated biological actions, including cell growth, migration and metabolism, but the intracellular signalling pathways leading to insulin‐induced cytoskeletal reorganization have largely been unknown. We therefore investigated the involvement of Grb2/Ash‐Ras and phosphatidylinositol (PI) 3‐kinase in the insulin‐induced morphological changes in fibroblasts over‐expressing human insulin receptors (HIRcB cells).

Insulin-independent and wortmannin-resistant targeting of IRS-3 to the plasma membrane via its pleckstrin homology domain mediates a different interaction with the insulin receptor from that of IRS-1.

Abstract.Aims/hypothesis: In primary adipocytes, although IRS-1 and IRS-3 are expressed in comparable amounts, these proteins manifest distinct distribution and significance in insulin signalling. We investigated the molecular basis of the difference between these two proteins. Methods: In Cos-1 cells transiently expressing rat IRS-1, IRS-3, or chimeric proteins of these two proteins we examined the tyrosine phosphorylation via the wild-type or mutant insulin receptors and evaluated their targeting to the plasma membrane by immunostaining the membrane ghost. Results: In contrast to IRS-1, IRS-3 was tyrosine-phosphorylated by the insulin receptor altering Tyr960 to Phe (Y960F), which disrupts the binding site of the PTB domain of IRSs, to an extent comparable to the wild-type receptor. The tyrosine phosphorylation of IRS-3 with the PH domain replacement via the Y960F insulin receptor markedly decreased, whereas that of IRS-3 with the PTB domain alteration was mildly impaired. Insulin-stimulated translocation of IRS-1 to the plasma membrane, as well as that of IRS-3 with the PH domain replacement, was wortmannin-sensitive, although that of IRS-3 was insulin-independent and wortmannin-resistant. Conclusions/interpretation: The affinity of the PH domain for the phospholipids in the plasma membrane seems to influence the receptor-substrate interaction required for IRS tyrosine phosphorylation, indicating that the PH domain and the PTB domain of IRSs cooperatively function in insulin-stimulated tyrosine phosphorylation of these proteins. [Diabetologia (2001) 44: 992–1004]